Fluorescence microscopy is one of the most extensively used tools for the structural and functional investigation of the interior of cells. Its popularity has steadily grown despite the fact that it notoriously fails to image structures smaller than about half the wavelength of light (˜200 nm), i. e. that it is limited by the so called diffraction barrier. While electron, X-ray, and scanning probe microscopy offer a substantially better spatial resolution, they all fall short in imaging intact or even living cells in three dimensions (3D). The invention of Stimulated Emission Depletion Microscopy (STED) in 1994 highlighted the then unexpected fact that the diffraction barrier to the spatial resolution can be effectively overcome in a microscope that uses regular lenses and focuses visible light [1,2]. Other subdiffraction resolution techniques, such as PALM, STORM and structured illumination have since emerged as well [3-5]. STED microscopy currently provides nanometer scale resolution [6-8] in biological and non-biological samples, while retaining most of the advantages of far-field optical operation, such as the ability to non-invasively image cells in 3D [9].
While the principles of scanning STED microscopy do not rest on those of the confocal microscope, STED can be implemented in a scanning confocal microscope to great effect. To this end, one overlaps the focused excitation beam of a scanning (confocal) microscope with a donut-shaped STED beam [10,11], whose role is to keep fluorophores dark even when they are exposed to excitation photons, and which is a particular embodiment of the beam of suppression light referred to above. The fluorophores remain dark, because the wavelength and the intensity of the STED beam are adjusted such as to instantly de-excite potentially excited fluorophores by stimulated emission. Consequently, fluorophores subject to a STED beam of intensity I>3 IS are practically confined to the ground state and hence switched off. This is a consequence of the fact that the normalized probability of the molecule to spend time in the excited state follows ˜exp(−I/IS), with IS being a characteristic of the molecule. Any molecule subject to I>>IS is deprived of its ability to fluoresce, because the fluorescent state is disallowed by the presence of the STED beam. Since I increases from the center of the donut on outwards to the donut crest, the probability for a molecule to be off is highest at the donut crest. Molecules located at the donut center remain fluorescent. At a certain distance from the center where I>3 IS, practically all molecules (95%) will be off. Since the threshold 3 IS can be moved towards the center by increasing the overall intensity of the STED-beam, the region in which the fluorophores are still capable of signalling can be decreased far below the physical width of the donut minimum, i.e. far below the diffraction barrier.
Specifically, for a wavelength λ and a numerical aperture NA of the objective lens, the spot in which the fluorophores are able to signal will have a diameter d≈λ(2 NA (1+Im/IS)1/2) [7,12]. Im is the intensity of the STED-beam at the donut crest. IS is usually of the order of 1 to 10 MW/cm2. Scanning the two overlapped beam reveals structures at a spatial resolution of d, because the signal of fluorophores that are further apart than d are recorded sequentially in time. With several current dyes, d can thus be shrunk down to ˜20 nm [6,7]; for a certain class of inorganic fluorophores (crystal color centers) even 5.8 nm have been reported [8].
An important point in setting up and operating a STED microscope is beam alignment. For maximum performance, the donut should be centered on the excitation spot with deviations <50 nm. Furthermore, the beam alignment should be stable over the course of a measurement and over an adequate field of view. While this is not an obstacle in principle, given that in standard multi-color confocal microscopes several beams are superimposed with a comparable precision, too, it is desirable to improve stability and ease of operation by having pre-aligned beams. Intrinsic alignment can be achieved by using a common laser source for both the excitation and the depletion beam. This can be accomplished by coupling two separate lasers into a common optical fiber or, even more conveniently, by using a super-continuum light source [13]. However, having pre-aligned beams requires a beam shaping device that leaves the excitation wavelength unaffected, while treating the STED wavelength in such a way that it forms a donut. Current donut-shaping devices however use a vortex phase mask and cannot sufficiently distinguish between wavelengths. They also forge the excitation beam into something close to a donut and are thus not suitable for the use with pre-combined beams. The solution suggested in [14] relies on the annular separation of pre-aligned beams but blocks a considerable amount of STED light. The method proposed in [15] has, up our knowledge, not been realized in practice so far.
More recently, Wildanger et al. [16] proposed a scheme that relies on the different dispersion properties of different optical materials. By selecting two optical glasses whose refractive indices match at the excitation wavelength but differ for the STED wavelength, they were able to design a phase plate that can be shared by both beams. In this scheme, however, the detection beam path is coupled out between the objective lens and the phase plate using a dichroic mirror.
In general, the same points listed above as relevant to STED microscopy also apply to GSD (Ground State Depletion) microscopy. In GSD microscopy the beam of suppression or depletion light depletes the ground state of the fluorophore out of which it is excitable for fluorescence by the excitation light in that the fluorophore is transferred into a dark state which can be a triplet state for example.
International Patent Application Publication WO 2008/145371 A2 proposes an optical assembly for use in STED and GSD microscopy which comprises an objective for projecting two optically different light components into a projection space, and an optical component that selectively deforms wave fronts of one of the light components passing through such that the intensity distribution of the one of the light components in the projection space, due to interference with itself, differs from the intensity distribution of the corresponding other light component in the projection space, the wave fronts of both light components as well as light emitted out of the projection space and collected by the objective passing through the optical component. The two light components may differ in wavelength and/or polarization.